This project is aimed to understand the molecular mechanisms by which calmodulin (CaM) recognizes and modulates the enzymatic activity of myosin light chain kinase (MLCK). Specifically this project is focused on the following problems: (1) Conformational changes induced in CaM by Ca2+- binding and by interaction with MLCK. Changes in spatial relation between helical segments in each of the two domains of CaM and changes in the interdomain distance will be studied. Mutants of CaM will be synthesized with Cys residues in suitable positions and changes in distances between the Cys side chains will be monitored by resonance energy transfer. (2) Functional significance of conformational transitions in CaM. Mutants of CaM will be designed in which movement of selected protein segments will be restricted by introduction of disulfide bonds. The ability of such mutants to bind MLCK and to modulate its function will be evaluated. (3) Definition of the sites of interaction with MLCK in CaM. The contribution of hydrophobic and ionic groups to the interaction will be assessed. By monitoring the regulatory ability of the CaM mutants we will distinguish the structural features that are essential for binding to MLCK from those that are involved in modulation of function. (4) Conformational transitions in MLCK in relation to its activation by CaM. We will use mutants of MLCK with cysteine residues at selected positions and monitor changes in their separation by resonance energy transfer. Probes will be placed in regions corresponding to the CaM-binding site, the pseudosubstrate region, the catalytic site and the interaction site with the light chain of myosin. distances between these sites and the natural landmarks in MLCK will be measured. (5) Functional significance of the N- terminal region of MLCK. A series of mutants of MLCK with systematic truncation from the N-terminus will be prepared, and their ability to interact with the regulatory light chain will be assayed, both alone and when complexed with the heavy chain of smooth muscle myosin.